An aneurysm is an abnormal dilation of an artery that, if left untreated, can lead to fatal tear or rupture. TGF? signaling upregulation is observed in aortic tissue from patients affected by hereditary aneurysm disorders, such as Loeys-Dietz syndrome (LDS), that are caused by heterozygous loss-of-function mutations in genes encoding positive regulators of the TGF? pathway. These paradoxical findings have caused considerable controversy and have called into question the use of TGF? antagonists as therapy for these disorders. The goal of this proposal is to identify the cellular and biochemical mechanisms that cause TGF? signaling overdrive in LDS. We propose to test a non-cell autonomous mechanism based on the observation that LDS mutations in either of the two genes that encode TGF? receptor subunits have discordant effects on the signaling capacity of aortic cell types that are adjacent in the vessel wall but derived from different embryonic progenitors. Specifically, these mutations leave high responder cells such as cardiac neural crest-derived vascular smooth muscle cells (CNC-VSMCs) relatively unaffected, while they impair the TGF? response of more vulnerable low responder cells, such as second heart field-derived VSMCs (SHF-VSMCs). The overall hypothesis of this proposal is that compensatory events aimed at restoring normal signaling in low responder cells, such as upregulation of TGF? ligand expression, will induce TGF? signaling overdrive in high responder cells, ultimately culminating in the development of aneurysm. In order to test this hypothesis, I will work with my primary mentor, Dr. Dietz, an expert on aneurysm pathogenesis, and with my co-mentor, Dr. Van Eyk, an expert in clinical proteomics applications, to address the first two aims. In Aim1, I will define the cellular mediators of TGF? paracrine overdrive by analyzing the TGF? response of control and LDS SHF-VSMCs and CNC-VSMCs, and the repertoire of paracrine factors secreted by these cells both in vivo and in vitro. In Aim 2, I will functionally test the TGF? paracrine overdrive model by pharmacological and genetic manipulations. Specifically, I will assess whether global TGF? antagonism or cellspecific reduction of TGF? signaling in high responder cells, but not low responder cells, ameliorates aneurysm progression in LDS mice. During this phase of the award I will become proficient in the field of proteomics and bioinformatics by working with Dr. Van Eyk's lab and by participating in formal coursework. The new skills, techniques, and knowledge that I will gain will foster my transition to the R00 phase, in which I will independentl address Aim 3 and Aim 4. In Aim 3 I propose to identify the molecular determinants of lineagespecific TGF? responses by using proteomics-based techniques and conventional biochemical methods to analyze, both in vitro and in vivo, the differential expression and/or post-translational modifications of modulators of TGF? signaling in high responder and low responder cells. In Aim 4, I intend to elucidate the contribution of CD45+ immune cells to TGF? signaling paracrine overdrive. I will characterize the phenotype of the CD45+ immune cells infiltrate that is present at the site of aneurysm in LDS mouse models and assess whether TGF? ligand secreted by these cells contributes to exacerbation of pathology. I am confident that the training and career development plan described in this proposal will enable me to elucidate the mechanisms of excessive TGF? signaling in LDS and will provide critical information for the understanding of cell-type specific TGF?-responses. The studies described in this proposal have the potential to increase our understanding of other TGF?-related disorders, such as fibrosis and cancer, in which signaling imbalances between interacting cell-types may drive pathology.